REVIEW ARTICLE
Standard methods for molecular research in
Apis
mellifera
Jay D Evans
1*, Ryan S Schwarz
1, Yan Ping Chen
1, Giles Budge
2, Robert S Cornman
1, Pilar De la Rua
3,
Joachim R de Miranda
4, Sylvain Foret
5, Leonard Foster
6, Laurent Gauthier
7, Elke Genersch
8,
Sebastian Gisder
8, Antje Jarosch
9, Robert Kucharski
5, Dawn Lopez
1, Cheng Man Lun
10,
Robin F A Moritz
9, Ryszard Maleszka
5, Irene Muñoz
3and M Alice Pinto
111USDA-ARS, Bee Research Lab, BARC-E Bldg 306, Beltsville MD 20705, USA.
2National Bee Unit, Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK. 3Department of Zoology and Physical Anthropology, Faculty of Veterinary, University of Murcia, Spain. 4Department of Ecology, Swedish University of Agricultural Sciences, Uppsala, 750-07, Sweden.
5Research School of Biology, The Australian National University, Australia Australian National Univ. Canberra, ACT 0200,
Australia.
6Centre for High Throughput Biology, University of British Columbia, Vancouver, BC, Canada.
7Swiss Bee Research Centre, Agroscope Liebefeld-Posieux Research Station ALP, Bern, CH-3003, Switzerland. 8Institute for Bee Research, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany.
9Martin-Luther-University Halle-Wittenberg, Dept. of Biology, Molecular Ecology, Hoher Weg 4, 06099 Halle, Germany. 10George Washington University, Department of Biological Sciences, Lisner Hall 340, 20234 G St. NW, Washington, DC 20052,
USA.
11Mountain Research Centre (CIMO), Polytechnic Institute of Bragança, Bragança, Portugal.
Received 19 March 2012, accepted subject to revision 25 September 2012, accepted for publication 11 February 2013. *Corresponding author: Email: [email protected]
Summary
From studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al. (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees:
Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.
Footnote: Please cite this paper as: EVANS, J D; SCHWARZ, R S; CHEN, Y P; BUDGE, G; CORNMAN, R S; DE LA RUA, P; DE MIRANDA, J R; FORET, S; FOSTER, L;
GAUTHIER, L; GENERSCH, E; GISDER, S; JAROSCH, A; KUCHARSKI, R; LOPEZ, D; LUN, C M; MORITZ, R F A; MALESZKA, R; MUÑOZ, I; PINTO, M A (2013)
Standard methodologies for molecular research in Apis mellifera. In V Dietemann; J D Ellis; P Neumann (Eds) The COLOSS BEEBOOK, Volume I: standard methods
Métodos estándar para la investigación molecular en
Apis
mellifera
Resumen
Las abejas de miel han sido durante mucho tiempo un organismo clave para avances fundamentales en biología a partir de estudios de su comportamiento, comunicación química, genómica y de biología del desarrollo, entre otros muchos. Con la secuencia del genoma en la mano y herramientas genéticas mucho mejores, las abejas son ahora un blanco aún más atractivo para responder a las preguntas más importantes de la biología evolutiva, la estructura de las poblaciones y la organización social. Al mismo tiempo, los incentivos agrícolas para entender cómo las abejas caen enfermas, o evadir y sobrevivir a sus muchas plagas y patógenos, han presionado para comprender genéticamente la inmunidad individual y social en esta especie. A continuación se describen y se hace referencia a herramientas que hacen uso de modernas técnicas de biología molecular para entender el comportamiento de las abejas, su salud y otros aspectos de su biología. Nos centramos en las técnicas de ADN y ARN, en gran parte debido a que las técnicas de evaluación de las proteínas de la abeja se tratan en detalle en Hartfelder et al. (2013). Cubrimos las necesidades prácticas de toma de muestras de abejas, su transporte y almacenamiento, y luego se discuten una serie de técnicas actuales de análisis genético. A continuación, se proporciona una hoja de ruta para los recursos genómicos y métodos para estudiar las abejas, seguido de protocolos estadísticos específicos de la genética de poblaciones, la genética cuantitativa y la filogenia. Finalmente, se termina con tres herramientas importantes para predecir la regulación génica y la función en las abejas melíferas: la hibridación in situ fluorescente (FISH), la interferencia de ARN (iARN), y la estimación de la metilación cromosómica y su papel en la regulación epigenética de los genes.
西方蜜蜂分子研究的标准方法
摘要
通过行为、化学通讯、基因组和发育生物学等方面的研究，蜜蜂已经成为用于在生物学基础研究领域取得重大突破的一种重要模式生物。结合已 有的基因组序列和多种改进的遗传学工具，蜜蜂已经越加成为回答进化生物学、种群结构和社会性结构等方面重大问题极具吸引力的研究目标。 与此同时，农业上为了解蜜蜂如何困于病害或者避开和幸存于多种害虫和病原菌的危害，也促进了对这一物种个体和社会免疫的遗传学理解。以 下我们介绍和引用了一些运用现代分子生物学技术研究蜜蜂行为、健康、以及其它方面生物学的工具。Hartfelder等2013已对研究蜜蜂蛋白做了 详细的论述，因此我们将重点放在DNA和RNA技术上。本文也包含了在蜜蜂采样、运输和保存过程中的实际需要，并讨论了当前的一系列遗传分 析技术。然后我们提供了研究蜜蜂时所需的基因组资源和方法的路线图，以及群体遗传学、数量遗传学和系统发生学研究中特定的统计学方法。 最后，我们以预测蜜蜂基因调控和功能的三个重要工具收尾：荧光原位杂交（FISH）、RNA干扰（RNAi）和染色体甲基化及其在表观遗传基因 调控中作用的估算。Keywords: Apis mellifera, pollination, disease, development, genomic, Colony Collapse Disorder, population genetics, methylation, RNA
interference, RNAi, Southern Blot, Northern Blot, In situ Hybridization, DNA extraction, Next-generation sequencing, mitochondrial DNA, microsatellite, quantitative PCR, COLOSS, BEEBOOK, honey bee
Page No.
4. RNA methods 14
4.1. Introduction 14
4.2. Affinity column purification 14
4.3. Acid Phenol RNA extraction from adult bees 15
4.3.1. TRIzol extraction 15
4.3.2. Bulk Extraction of RNA from 50-100 whole bees using
the acid-phenol method 16
4.3.3. RNA lysis/stabilization buffer 17
4.4. RNA quality assessment 17
4.5. cDNA synthesis from total RNA 17
4.5.1. Reverse Transcription of RNA 17
4.6. Qualitative RT-PCR for honey bee and pathogen targets 18
4.7. Quantitative RT-PCR for honey bee and pathogen
targets 18
4.7.1. One-Step versus Two-Step RT-PCR 19
4.7.2. One-Step RT-qPCR 19
4.7.3. Two-Step RT-qPCR 19
4.7.4. Two-step Quantitative PCR for high-throughput assays 19
4.7.5. Multiplex RT-(q)PCR 20
4.8. Primer and probe design 20
4.8.1. Primer length, melting temperature and composition 20
4.8.2. Annealing temperature 20
4.8.3. Cycling parameters 21
4.9. Assay optimization 21
4.9.1. Primer-dimers and other PCR artefacts 21
4.9.2. Primer concentration 21
4.9.3. Magnesium concentration 21
4.10 Quantitation controls 21
4.10.1. External reference standards 21
4.10.2. Internal reference standards 23
4.10.2.1. Exogenous internal reference standards 23
4.10.2.2. Internal reference standards 23
4.10.2.3. External standard for viral target quantification 24
4.11. Microarrays 24
4.12. Northern blots using DIG labelling 24
4.12.1. Agarose / Formaldehyde gel electrophoresis 24
4.12.2. Assembly of the transfer setup and transfer of RNA
from gel to membrane 25
4.12.3. Preparation of DIG labelling (non-radioactive) probe 25
4.12.4. Hybridization analysis 26
4.13. In situ hybridization 26
4.13.1. Tissue fixation 26
4.13.2. Preparation of DIG labelling (non-radioactive) probe 26
4.13.3. Hybridization analysis 26
5. Proteomic methods 27
5.1. Introduction 27
6. Population genetics 27
Table of Contents
PageNo. 1. Sample management 5 1.1. Introduction 5 1.2. Sample collection 5 1.2.1. Adult bees 5 1.2.1.1. Nurse bees 5 1.2.1.2. Foraging bees 5 1.2.2. Pupae 5 1.2.3. Larvae 5 1.2.4. Eggs 5 1.2.5. Extracted guts 6
1.2.6. Drone endophalus and semen 6
1.2.7. Faeces 6
1.2.8. Dead bee samples 6
1.3. Sample transport 6
1.3.1. Freezing with dry ice 6
1.3.2. Freezing with “wet” ice 6
1.3.3. Live transport 7
1.3.4. Chemical stabilizers 7
1.3.5. Sample collection cards 8
1.4. Long-term sample storage 8
1.4.1. Freezing 8 1.4.2. Drying 8 1.4.3. Chemical stabilizers 8 2. Sample processing 9 2.1. Introduction 9 2.2. Sample homogenization 9 2.2.1. Bead-mill homogenizers 9 2.2.2. Blender 9 2.2.3. Paint shaker 9
2.2.4. Mortar and pestle 9
2.2.5. Mesh bags 10
2.2.6. Micropestle 10
2.2.7. Robotic extraction 10
3. DNA extraction and analysis 10
3.1. Introduction 10
3.2. Genomic DNA extraction from adult bees 10
3.2.1. DNA extraction using CTAB 10
3.2.2. DNA extraction using Qiagen Blood and Tissue DNA kits 12
3.2.3. DNA extraction using Chelex 12
3.3. DNA detection using southern blots with DIG labelling 12
3.3.1. Restriction enzyme digestion and agarose gel
electrophoresis 12
3.3.2. Assembly of the transfer setup and transfer of DNA
from gel to membrane 13
3.3.3. Synthesis of DIG-labelled DNA probe 13
3.3.4. Hybridizing the DIG-labelled DNA Probe to DNA on the
Table of Contents cont’d
Page No.6.1. Introduction 27
6.2. Mitochondrial DNA analysis 27
6.3. Nuclear DNA analysis 28
6.3.1. Microsatellites 28
6.3.1.1. Microsatellite reaction mix 28
6.3.1.2. Primers for multiplexed honey bee microsatellite loci 29
6.3.1.3. Thermal cycling conditions for multiplex PCR 29
6.3.1.4. Size estimation of PCR products 29
6.3.2. Single-nucleotide polymorphisms (SNPs) 29
7. Phylogenetic analysis of sequence data 30
7.1. Introduction 30
7.2. Obtaining and formatting sequences of interest for
phylogenetics 30
7.3. Sequence data in FASTA format 30
7.4. Alignment of sequence data 31
7.4.1. Clustal 31
7.4.2. MUSCLE 31
7.5. Trimming aligned sequence data to equal length 31
7.6. Performing phylogenetic analyses 31
7.6.1. Using MEGA 31
7.6.1.1. Converting data to MEGA format 32
7.6.1.2. Constructing and testing phylogenetic trees 32
7.6.2. Using SATé 33
7.6.2.1. External tools 33
7.6.2.2. Sequence import and tree building 33
7.6.2.3. Job Settings 33
7.6.2.4. SATé Settings 33
7.6.3. Building trees using distance and character based
methods 33
8. Genomic resources and tools 34
8.1. Introduction 34
8.2. Honey bee genome project 34
8.3. Honey bee parasite and pathogen genomes 34
8.4. Comparative genomics 34
8.5. Second-generation sequencing 35
8.6. Genomic sequence assembly 35
8.7. Transcriptomic analyses (“RNASeq”) 35
8.8. Metagenomics 35
8.8.1. RNA versus DNA sampling 35
8.8.2. Sample preparation 36
8.8.3. Amplicon-based or shotgun sequencing 36
Page No.
8.8.4. Assembly of shotgun sequences vs. read mapping 36
8.8.5. Databases for metagenomics 36
8.8.6. Post-assignment statistics 36
9. Fluorescence in situ hybridization (FISH)
analysis of tissues and cultured cells 37
9.1. Introduction 37
9.2. Tissue fixation and tissue sectioning exemplified with
gut tissue 37
9.3. Fixation of cultured cells grown in suspension 37
9.4. FISH-analysis of tissue sections and fixed insect cells 37
10. RNA Interference 38
10.1. Introduction 38
10.2. Production of RNA interfering molecules 38
10.2.1. siRNA design and synthesis 38
10.2.2. Production of dsRNA 39
10.3. RNAi Applications 40
10.3.1. RNAi in adult honey bees via feeding 40
10.3.2. RNAi in honey bee larvae via feeding 40
10.3.3. Gene knock-down by abdominal injection of
target-specific dsRNA/siRNA 40
10.4. Concluding remarks 40
11. DNA methylation in honey bees 41
11.1. Introduction 41
11.2. DNA methylation in honey bees 41
11.3. DNA extraction from various tissues for methylation
analysis 41
11.4. High-throughput sequencing of targeted regions 42
11.4.1. Fragmentation of DNA 42
11.4.2. End-repair of sheared DNA 42
11.4.3. Adaptor ligation 42
11.4.4. Size selection of adapter-ligated fragments 42
11.4.5. Bisulfite conversion and amplification of the final
library 42
11.4.6. Validation of the libraries 42
11.4.7. Sequencing and data analysis 42
11.5. Mapping and methylation assessment 42
11.6. Methylation dynamics and expression of individual
genes 43
11.6.1. Amplicon sequence selection 43
11.6.2. Bisulfite DNA conversion 43
11.6.3. Bisulfite PCR 43
11.7. RNA extraction 44
11.8. cDNA synthesis and template quantification 44
12. Acknowledgements 44
1. Sample management
1.1. Introduction
In order to best reflect honey bee biology, data generated from molecular-genetic studies should reflect as closely as possible the state of honey bee tissues, entire bees, or colonies just prior to sampling. This fact places a premium on collecting and storing samples in a way that retains this state. Although technological developments in molecular biology allow for a great diversity of insights from collected bee samples, it is often forgotten how much these insights are hampered by errors in the collection, storage and processing of samples (Chernesky et al., 2003). These problems are especially evident when data from different studies or laboratories are compared (Birch et al., 2004). The only solution to this is optimization of collection, storage and primary processing protocols, so as to minimize the influence of sample degradation on the molecular analyses and the reliability of the data. As is often the case, cues can be taken from other areas of biology, notably the medical field, where such practices are widely adopted (Valentine-Thon et al., 2001; Verkooyen et al., 2003).
A secondary consideration is that a sample may be used for several different analyses; proteins, nucleic acids, fats and lipids, metabolites etc., requiring a collection and processing protocol suitable for all compounds analysed. Usually this means that the sample management conditions follow the requirements for the least stable of the compounds, which for bee research is usually the RNA. RNA is highly sensitive to degradation by robust RNAse enzymes found in all cells, unless the sample is stabilized with RNAse-inhibiting additives and/or frozen as soon as possible. Given the necessity of RNA analyses for many questions related to bees and their parasites and pathogens (e.g., de Miranda et al. 2013), field-appropriate methods for stabilizing RNA are required.
1.2. Sample collection
The optimum strategy for collection and transport of bee samples depends partly on what type of sample is collected. Bees, pupae, larvae and eggs can be sampled whole or as field-dissected components, such as heads, thoraxes, abdomens, guts, endophalli, semen, ovaries etc. Many bee viruses are shed in large amounts in the guts, as are many bacterial and protozoan pathogens (Shimanuki, 1997; Fries, 1997). Faeces may therefore be a good marker for the infection status of the whole bee, although care has to be taken to distinguish between passively acquired/passaged microbes and true tissue infections. Faeces also allow bees to be sampled repeatedly, and non-destructively. It may therefore be useful for determining the virus status of queens (Hung, 2000), especially if these are a major source of infection of the worker population (Chen et al., 2005b; Fievet et al., 2006), or for following disease progression in individual bees.
Below are suggestions for the collection of different types of bee samples. In all cases a priori decisions are all needed with respect to the use of chemical stabilizers, collection cards and the temperatures during transport and storage.
1.2.1. Adult bees 1.2.1.1. Nurse bees
Inspect each frame in a colony and find a frame with sealed and unsealed brood which is covered by adhering nurse bees and then take the frame out of the colony.
1.2.1.2. Foraging bees
Block the hive entrance where foraging bees are accumulating and collect the returning foraging bees.
1.2.2. Pupae
1. Cut out a section of sealed brood, to be transported whole. Such a brood section can be sent through the post, although with the caveat that such transport away from the hive might affect bee or parasite gene activities.
2. Uncap brood cells, lift pupae by their neck by curling fine curved forceps underneath their heads and transfer to a suitable transport medium, either individual microcentrifuge tubes or collection cards (see section 1.3.5).
1.2.3. Larvae
1. Cut out a section of open brood and transport in a
temperature -humidity controlled box, to prevent dehydration. 2. Remove larvae from the comb using either a blunt grafting
needle (small larvae) or soft forceps (large larvae) and transfer to individual microcentrifuge tubes or collection cards.
1.2.4. Eggs
1. Cut out a section of comb with eggs and transport in a temperature-humidity controlled box, to prevent dehydration. 2. Remove eggs using a blunt needle and transfer individually or
in bulk to microcentrifuge tubes or collection cards. As an alternative for the rapid collection of massive amounts of eggs and early embryos:
1. Strike soundly a frame containing early-stage bees onto a sterile surface twice.
This releases over half of the eggs and embryos held by that frame, 2. Brush or lift into a new vessel.
While uncapped honey will drip via this method, if done at the right intensity, older uncapped larvae will remain in their cells.
1.2.5. Extracted guts
1. Grab the stinger and last integument of adult worker bees firmly with a pair of fine, straight forceps.
2. Pull backwards gently, removing the whole hindgut and midgut.
3. Transfer guts to individual microcentrifuge tubes or collection cards (see section 1.3.5.).
1.2.6. Drone endophallus and semen
1. Turn drone upside down and grip laterally between thumb and index finger.
2. With the other hand, gently but persistently squeeze the abdomen of the drone dorso-ventrally, exerting pressure backwards, until the endophallus is extruded from the drone. 3. Apply more severe pressure, again backwards, to avert the
endophallus and, for mature drones, cause ejaculation of semen.
4. Cut off the entire endophallus with scissors, or collect the exposed semen (brown-red colour) and/or seminal fluid (translucent white) with a sterile micropipette.
5. Collect the material individually in microcentrifuge tubes or on collection cards.
1.2.7. Faeces
If destructive sampling is allowed:
1. Remove the whole gut from individual bees (see above) and expel faecal mass.
If repeated sampling is required:
1. Place adult bees into a Petri dish until defecation has occurred.
2. Collect faeces individually or pooled in microcentrifuge tubes or on collection cards.
1.2.8. Dead bee samples
Many bee disease experiments involve bee death as a parameter. Dead bee samples from such experiments are, of course, valid material for analysis. They should be treated like freshly killed material and frozen as soon as possible to minimize the effects of decay on RNA integrity, using the collection methods appropriate for the sample type, as given above. Dead bee traps attached to hives are suitable for collecting such bees and should be emptied daily to minimize the effects of decomposition.
Passive surveys also involve dead bee samples, in this case those sent in by beekeepers for post-mortem analysis of the cause of colony death. These bees will have been dead long enough for decomposition and drying to have severely affected the integrity of the RNA, including viral RNAs. Such degradation can severely affect the accuracy and reliability of detecting and quantifying individual RNAs (Bustin and Nolan, 2004; Fleige and Pfaffl, 2006; Becker et al., 2010). This means
that only positive results from such samples are informative, since negative results can be either due to the absence of virus or the degradation of the RNA.
It is possible to adjust for differential RNA degradation in the different samples with quantitative RT-qPCR techniques, by using host internal reference gene levels for normalizing the virus titers (Dainat et al., 2011) and setting the threshold for template detection with the most degraded sample, so that all samples are evaluated by the same degradation criteria. How to determine the detection thresholds using RT-qPCR assays is covered in section 4.4.
1.3. Sample transport
Sample transport from the collection site to the laboratory is the most critical step in sample management, since this is where the integrity of the sample is most easily compromised (Chen et al., 2007). Sample integrity can be preserved to different degrees with the following methods, given in order of effectiveness. The gold standard for sample collection and transport is to freeze on-site, but this is not always possible. All alternatives are basically aimed at getting the samples as quickly and conveniently as possible into a freezer, with minimum degradation. The most useful tool for transporting frozen material is a liquid nitrogen-based ‘dry shipper’, which is specifically developed and approved for international shipment of biological samples at ultra-low temperatures (-150oC). The best can hold these temperatures for
more than one week. Other options, for more local transport, are (dry) ice-boxes and portable/car freezers. Courier and mail services are less reliable, both with respect to the maintenance of temperature and the duration of transport.
1.3.1. Freezing with dry ice
Samples: all.
Conditions: freeze instantly; keep frozen throughout transport using blocks of dry ice in a cooler.
Transport: restricted transport; dry ice must be replenished ca. every 48 hours.
Processing: transfer samples to freezer. Pros: gold standard; fast.
Cons: very expensive; complex field operation.
1.3.2. Freezing with ‘wet’ ice
Short-term field-storage on ‘wet’ (frozen water or ice packs) ice is cheap and very practical for many field-studies and surveys. The samples should be frozen as soon as possible, ideally within hours, and kept frozen continuously until RNA processing (a complete frozen transport chain). If a complete frozen transport chain cannot be guaranteed, then a chemical stabilizing agent (see section 1.3.4.) should be used to prevent degradation of the RNA by RNAses, until the samples enter a frozen transport chain. The most important rule for RNA preservation is to keep the samples as cold as possible, as
long as possible and to avoid thawing the sample after it has been frozen unless it is to extract RNA.
Samples: all.
Conditions: collect in freezer bags, store on wet ice. Transport: cold transport; wet-ice; < 12 hours. Processing: transfer samples to freezer. Pros: simple; fast; cheap field operation.
Cons: heavy, expensive mail transport, leaks due to thawing.
1.3.3. Live transport
Bees can also be transported live, which allows them to be sent much more quickly, cheaply and reliably by post than frozen samples. One drawback is that the stress of live transport may affect the expression of host genes, and possibly virus replication, which should be taken into account when planning experiments.
1. Adult bees can be transported live 1) In a well-ventilated bee shipping box containing queen candy and a sponge soaked in water glued to the bottom of the box or 2) in units of 10-15 bees in commercial queen cages with queen candy. Such queen cages are readily available to most beekeepers. Samples: adults.
Conditions: room temperature. Transport: < 48 hours. Processing: freeze on arrival.
Pros: simple; fast; suitable for beekeepers. Cons: stress during transport.
2. Pupae can be transported live 1) as a section of capped brood in a well-ventilated bee shipping box, preferably in a warm environment to prevent chilling, 2) as queen cells for queen pupae in a specialized temperature-humidity controlled queen-cell transport container, available from beekeeping suppliers. Such cells should be handled with great care, as developing queen pupae are very sensitive to disturbance, or 3) as a whole frame in a specialized carrier box for frames, available from beekeeping suppliers, or in a swarm box/nucleus hive. Samples: pupae.
Conditions: room temperature. Transport: < 48 hours.
Processing: remove samples from comb and freeze. Pros: simple, fast.
Cons: pupae may emerge during transport.
3. Larvae and eggs can be transported live 1) as a section of comb, in a temperature and humidity-controlled box or 2) as a whole frame in a specialized carrier box for frames, or in a swarm box/nuc.
Samples: larvae; eggs.
Conditions: controlled temperature and humidity. Transport: less than 48 hours.
Processing: remove samples from comb and freeze. Pros: simple, fast.
Cons: expensive by mail, unsealed larvae are subject to
temperature stress and starvation. 1.3.4. Chemical stabilizers
There are a number of chemicals that can be used to help stabilize nucleic acids during transport. Their purpose is to inhibit nucleases, especially the resilient RNAses, and in doing so destroy all enzymatic activity in the sample. So, if the final assays include natural enzymatic activity, these stabilizers should be avoided. For similar reasons, many stabilizers are also incompatible with serological detection methods, such as ELISA.
A large excess (5-fold by weight) of stabilizer should be added to ensure a high enough concentration within the tissues for inhibiting RNAses. It is also essential that the solution penetrates the tissues completely to abolish all RNAse activity. This is a major difficulty for aqueous stabilizers, which cannot penetrate the hydrophobic insect exoskeleton. These are therefore only suitable for extracted tissues, eggs and small larvae, unless bodies are partially disrupted at the start. Organic preservatives, such as 100% ethanol, have much more effective penetration of the exoskeleton and are therefore better for stabilizing whole adult bee samples. Although 100% ethanol is suitable for preserving RNA destined for short-fragment RT-qPCR-based assays, storage in 70% ethanol has been shown to result in strong degradation (Chen et al., 2007). However, recent data using a short amplicon (124 bp) diagnostic for Deformed wing virus (DWV) in a Taqman assay (Chantawannakul et al., 2006), showed no loss of DWV signal after adult bees were stored for 4 weeks in 70% EtOH at room temperature compared to snap frozen controls (G. Budge, unpublished data). RNA can also be stabilized by high concentration sulphate salt solutions (Mutter et al., 2004), of which RNAlater®
(Qiagen) is the best known. A generic version can be made as follows:
700 g di-ammonium sulfate 40 ml 0.5M EDTA (pH 8.0)
25 ml 1M tri-sodium citrate (di-hydrate salt; 29.4 g/100 ml) 1l sterile water
~1.3 l total volume
Once stabilized, RNase activities will be inhibited and samples can be stored for up to 1 month at 4°C, and long-term at -20°C or -80ºC with minimal degradation. The stabilizer should be removed from the bee sample prior to homogenization and RNA extraction.
1. 100% ethanol
Samples: whole adult bees; pupae; large larvae; tissues. Use: 5 volumes by weight.
Storage: 1 month at room temperature or lower.
Processing: Remove ethanol and process samples as normal. Pros: Cheap; effective penetration.
Cons: Evaporation; possible transport restrictions; heavy; incompatible with serological assays .
2. RNAlater® & generic equivalent
Samples: tissues; eggs; small larvae. Use: 5 volumes by weight.
Storage: 1 month at room temperature, or lower.
Processing: Remove stabilizer and process samples as normal. Pros: Non-hazardous; effective penetration.
Cons: Expensive (except generic version); heavy. It is possible to use RNAlater® for darkened pupae and adult
bees, if they are crushed into a paste or cut into 5mm sections (Chen et al., 2007). This is laborious and risks losing virus particles and RNA to the stabilizing solution, but may be required in certain circumstances. In such cases, the crushed bees should be centrifuged at 1,000 rpm for 5 minutes at 4oC before removing the stabilizer and processing the
crushed bee tissues.
1.3.5. Sample collection cards
Samples can also be dried on filter paper-based collection cards. In this case the molecules are stabilized primarily through desiccation, rather than low temperature, so thorough drying during sampling and low humidity during storage is essential for this method. The FTA™-cards produced by Whatman are furthermore impregnated with chemicals to prevent bacterial or enzymatic degradation of nucleic acids (Becker et al., 2004; Rensen et al., 2005). The method is ideal for liquid samples (blood, urine, sputum etc.) but has also been used for insect samples (Harvey, 2005) including honey bee larvae, pupae, extracted tissues and mites. Such filter-dried samples can be analysed for all manner of compounds (Jansson et al., 2003; Chamoles et al., 2004; Li et al., 2005; Zurfluh et al., 2005) including RNA (Karlson et al., 2003; Prado et al., 2005). The major advantages are the ease and reduced costs of collection, transport, labelling and long-term storage at room temperature, reducing the requirements for freezer space, boxes and tubes (Kiatpathomchai et al., 2004; Harvey, 2005; Karlson et al., 2003; Rensen et al., 2005; Prado et al., 2005). The major disadvantages are the uneven distribution of target across the filter paper and the gradual loss of target during prolonged storage (Chaisomchit et al., 2005). Samples collected on collection cards should therefore also be processed as soon as possible, by cutting out the entire dried sample and soaking this in an appropriate buffer, as recommended by the FTA™-card protocol, for the recovery of nucleic acids.
FTA™ collection cards
Samples: Tissues; faeces; eggs; larvae; pupae; mites. Use: Squash sample on card and air-dry.
Storage: At room temperature in dry container. Not in freezer. Processing: Cut out sample and soak directly in extraction
buffer for 15 minutes. Proceed as for fresh samples. Pros: Excellent preservation; light; easy storage and
indexing; versatile; preservation of faeces.
Cons: Expensive; variable processing; uneven distribution across card; not suitable for adult bees, not suitable for bulk samples.
1.4. Long-term sample storage
The critical factors for long-term sample preservation, as with degradation in the weeks after collecting, are minimizing the activity of nucleases. This can be achieved by a combination of:
1.4.1. Freezing
Freezing at -80ºC is the gold-standard for long-term storage of bee samples intended for RNA analysis. Freezing at -20ºC also provides good storage for preserving the quality of bee samples. However, significant to complete degradation of RNA can occur within days in dead bees kept at 4°C (Chen et al., 2007; Dainat et al., 2011). It is therefore strongly recommended to transfer frozen bees to the -80°C freezer immediately after samples are brought back from the field to the laboratory, if analysis is not initiated immediately.
1.4.2. Drying
Apart from drying soft bee stages and tissues on collection cards, bulk samples of whole bees can also be freeze-dried, or lyophilized. Lyophilization is a convenient way to store samples long-term at room temperature and preserves the chemical integrity of most compounds, although some functional activity may well be lost. Freeze-drying/ lyophilization requires a specialized instrument that draws a vacuum while the samples are kept below the point where solid and liquid phases can co-exist (below -50oC), so that the ice sublimates, i.e.
changes directly to vapour without melting first. Any biological sample can be lyophilized and the instructions for this come with the particular lyophilizing apparatus. It should be noted here that reconstituted dried tissue is fundamentally different from frozen wet tissue, with different and more variable recovery efficiencies for the different biomolecules than for fresh tissues. Lyophilized samples are stored at room temperature in a sealed box with desiccating packages, to prevent re-hydration.
1.4.3. Chemical stabilizers
There are several chemical agents that inhibit RNAses and thus reduce RNA degradation during handling and storage (see Section 4.4.4.). They do not provide any additional benefit to frozen samples,
2.2.2. Blender
An excellent, cheap alternative to the beadmills, especially for large volumes is homogenisation with a blender.
Pros: Large volume; rapid; uniform.
Cons: Cross-contamination risk due to re-use of blender; incompatible with organic solvents; corrosion of blender due to salts.
1. Add between 30-200 frozen bees to blender. 2. Add 500 μl ice-cold buffer per bee.
3. Homogenise by gradually raising the blender settings, for about 5 minutes total homogenization.
2.2.3. Paint shaker
A paint shaker (e.g. Automix shaker; Merris Engineering ltd) is a surprisingly efficient method of grinding bulk samples which has been used for various matrices including soil , grains, rice, wheat, honey, and bees (Woodhall et al., 2012; Budge, unpublished data). The method is completely scalable ranging using polypropylene wide-mouth environmental bottles (Nalgene) ranging in size from 60 ml to 2000 ml.
Pros: Large volume; easy; cheap; no cross-contamination; high throughput.
Cons: Large piece of equipment required.
1. Place 30-1000 frozen bees in an appropriately sized bottle (Nalgene) containing 5 x 25.4 mm stainless steel ball bearings.
2. Dry grind on the paint shaker for 8 minutes until the sample is sufficiently disrupted.
3. Add the required volume of extraction buffer depending on the protocol.
4. The addition of 1% Antifoam B (Sigma) to GITC, GHCl or CTAB extraction buffers can aide buffer recovery and reduce cross contamination.
5. Wet grind for a further 4 minutes. 6. Spin at 6000 g for 5 mins. 7. Recover supernatant.
2.2.4. Mortar and pestle
Traditional manual method for pulverizing samples. Pros: Medium volumes; cheap; low maintenance. Cons: Cross-contamination risk; time consuming; lack of
uniformity.
1. Place 1-30 bees in a pre-frozen mortar of appropriate size. 2. Add liquid nitrogen to cool samples to well below freezing. but can be useful for storing samples temporarily at room temperature.
Their effectiveness varies and they do not prevent degradation absolutely (Chen et al., 2007) but they are useful if minor degradation can be tolerated and the samples can be processed within a few months of collection.
2. Sample processing
2.1. Introduction
The initial processing of a sample is another key step in ensuring the uniformity and reliability of an assay. Nevertheless, generally little attention is paid to optimizing this part of the protocol for both maximum recovery of the target molecule(s) and for reducing variability. In general, the shorter and faster the protocol the better, since each additional step will contribute to the overall error and reduce the recovery efficiency, both of which compromise results. Here we will describe generalities of sample processing before independent chapters describing RNA and DNA extraction from samples.
2.2. Sample homogenisation
A highly variable step in sample processing is sample homogenization, not only between different homogenization options but also between different samples using the same protocol. The choice of
homogenization method depends on the sample type and number of bees per sample. There are numerous options outlined below.
2.2.1. Bead-mill homogenizers
These are the best option for uniform and reproducible homogenization of small (individual) bee samples. The samples are mixed with glass, ceramic or steel 1-3 mm beads and extraction buffer in sturdy disposable plastic tubes and shaken at high velocity in a machine. They also provide consistent cell wall disruption of bacteria and other microbes for parasite/pathogen or microbiome surveys.
Pros: Low-medium volume; rapid; uniform; no cross-contamination.
Cons: Generally only suitable for small bee samples (1-10 bees).
1. Place single bee in a 2 ml screw-cap microcentrifuge tube. 2. Add four 2 mm glass beads.
3. Add 500 μl ice-cold buffer.
For medium-large volume beadmills, increase the number of bees, beads and buffer in proportion to the maximum allowable volume of the disposable container.
4. Make sure that the bead mill is balanced, if this is a requirement. 5. Shake for 5-10 minutes at the highest setting.
3. Grind the bees to a powder using an appropriate sized, pre-frozen pestle.
4. Transfer the powder to a plastic tube or bottle. 5. Add 500 μl extraction buffer per bee.
6. Shake tube until the powder has suspended fully in the buffer.
2.2.5. Mesh bags
Mesh bags are sturdy plastic bags with a small pore fine mesh inside. The sample is placed on one side of the mesh, ground from the outside and the homogenate is collected from the other side of the mesh, filtering out large particles.
Pros: Medium-large volume; easy; cheap; no cross-contamination.
Cons: Lack of uniformity; split bags.
1. Place up to 30 frozen bees in a disposable mesh-bag (e.g., www.bioreba.com; #430100).
2. Add 500 μl buffer per bee.
3. Flash-freeze the entire bag in liquid nitrogen. 4. Remove from liquid nitrogen.
5. Wait 30 seconds.
6. Pulverize contents by grinding the bag with a large pestle for 2 minutes on a hard surface, taking care not to damage the bag.
7. Massage the bag until completely thawed.
8. Remove one (or more) 100 μl aliquots of homogenate. 9. As an alternate method, described in section 4.3.2. samples can
be crushed in disposable mesh bags using a heavy rolling pin.
2.2.6. Micropestle
You will need individual disposable micro pestles that fit
microcentrifuge tubes. These can be bought or made by heating a 1000 μl blue tip in a flame and moulding it into a disposable pestle in a microcentrifuge tube while it cools.
Pros: Single bees; cheap; low maintenance. Cons: Time consuming; lack of uniformity.
1. Grind a frozen bee tissue or larval sample with the micropestle in a microcentrifuge tube.
2. Discard pestle.
3. Add 500 μl buffer per bee. 4. Mix with a vortex.
2.2.7. Robotic extraction
Companies produce robotic extraction stations to facilitate high-throughput analysis of samples. Comparisons between several such systems, or between automated and manual extraction, generally find
little difference in terms of assay sensitivity and reproducibility (Rimmer et al., 2012; Bruun-Rasmussen et al., 2009; Agüero et al., 2007; Petrich et al., 2006; Knepp et al., 2003, but see Schuurman et al., 2005). Such systems are generally only suitable for easily disrupted, soft tissues or samples.
Pros: Single bees; rapid; high throughput; uniform; low cross contamination risk.
Cons: Expensive; inflexible protocols; soft tissues only. Follow manufacturers’ protocol for sample processing.
3. DNA extraction and analysis
3.1. Introduction
Isolating and analysing an organism’s DNA is key for developing insights into species or strain identification, for uncovering variants useful in breeding or a more thorough understanding of biology, and for discovering the microbes carried by individuals. DNA extraction methods must be robust for small amounts of starting material even if that material has become degraded. They must deliver extracted DNA of sufficient quality, purity, and quantity for downstream efforts ranging from target identification (e.g., via the Polymerase Chain Reaction, PCR, below in section 6.3.1.), sequence analysis, and cloning, among others. Below are tested protocols for common DNA analyses of diverse bee samples, starting with the isolation and purification of DNA. Isolating DNA from tissues can be accomplished using a variety of commercial kits, or via procedures built on standard disrupting and separating agents as below. Here we describe protocols made from primary ingredients, since this is illustrative of the critical components in these and pre-made extraction protocols.
3.2. Genomic DNA extraction from adult bees
3.2.1. DNA extraction using CTAB
This protocol is for the extraction of DNA from bee abdomens and/or the thorax, using a lysis buffer containing CTAB, a compound that is able to separate polysaccharides from other cell materials. The choice of tissues avoids eye contaminants such as pigments, which can inhibit PCR and other downstream applications. The method can be scaled down for the extraction of Varroa destructor mites (see the BEEBOOK paper on varroa (Dietemann et al., 2013) for details on sampling) or bee embryos and up for larger larvae and pupae (see section 1.2. for their collection). Volumes should be adjusted accordingly based on sample volume (i.e. initial grinding in 5X sample volume of buffer, ca.25-> 200 µl). The subsequent two extraction protocols are simpler, but the CTAB procedure is excellent for problematic samples and is flexible in terms of tissue disruption, separation, and rescue of nucleic acids.
1. Extract only the abdomen and/or thorax if possible. If a whole animal is extracted, use a Qiagen or similar column following manufacturer’s protocol for final purification of extracted DNA in order to reduce pigments that can inhibit genetic assays. 2. Put tissue from a single bee in a 1.5 ml microcentrifuge tube. 3. Add 500 µl of CTAB + 2 µl 2-mercaptoethanol (0.2%). CTAB buffer:
100 mM Tris-HCl, pH 8.0 1.4 M NaCl
20 mM EDTA
2% w/v hexadecyl-trimethyl-ammonium bromide (CTAB) This buffer both stabilizes nucleic acids and aids in the separation of organic molecules. See MSDS as CTAB is a potential acute hazard.
4. Homogenize with pestle.
5. Add 50 µg proteinase K and 25 µl of RNase cocktail. While this step is optional, proteinase K improves yields by disrupting cell and organelle boundaries and is critical for extraction of DNA from many microbes.
6. Vortex briefly to mix.
7. Incubate at 55-65°C from several hours to overnight. Invert occasionally during incubation (e.g. once every 30 minutes for the first two hours).
8. Centrifuge for 1 min at maximum speed (~14,000 rpm). Unwanted tissue debris will form a pellet at the bottom of the microcentrifuge tube.
9. Transfer liquid to fresh tube, leaving tissue debris pellet behind.
10. Add equal volume phenol:chloroform:isoamyl alcohol (25:24:1).
11. Invert several times (10-20 times) to mix then put on ice for 2 min.
12. Spin at full speed (~14,000 rpms) for 15 min at 4°C. 13. Transfer upper phase to fresh tube.
14. Add 500µl cold isopropanol + 50 µl 3 M NaOAc. 15. Vortex to mix, then incubate at 4°C > 30 min. Samples can be stored at ambient temperature at this point for several days if needed for transport or timing, otherwise 4°C is best.
16. Spin at full speed (~14,000 rpms) for 30 min at 4°C. 17. Carefully decant liquid from DNA pellet.
18. Add 1 ml 4°C 75% EtOH. Tap vortex briefly to loosen pellet. 19. Spin at full speed for 3 min at 4°C.
20. Decant liquid from pellet.
21. Air dry pellet about 10 minutes to evaporate all residual traces of alcohol.
Do not over dry pellet, as it will be hard to resuspend.
22. Resuspend in 50-100 µl nuclease-free water (overnight at 4°C). 23. Check DNA quantity and integrity on an agarose gel. 24. First, prepare TBE gel buffer (an aqueous solution with a final
working concentration of 45 mM Tris-borate and 1 mM EDTA). This is often prepared first as a ‘5x’ concentration comprised of 4 g Tris base (FW = 121.14) and 27.5 g boric acid (FW = 61.83) dissolved into approximately 900 ml deionized water. Add 20 ml of 0.5 M EDTA (pH 8.0) to this solution and adjust the solution to a final volume of 1l. Confusingly, the ‘working solution’ of this buffer for most uses is as 0.5x = a 1/10 dilution of the stock buffer.
25. For a 1.5% agarose gel on a large-format gel rig, add 3 g of sterile agarose to 200 ml TBE buffer in a 500 ml or larger
Erlenmayer flask, microwave at high heat for ca. 45 s (without boiling). For smaller gel rigs the volume of the gel can be from 50 to 100 ml. Take flask out and swirl, then heat in the microwave again until at full boil for 45 seconds, monitoring to avoid spillover. The agarose must fully dissolve so the liquid is perfectly clear
26. Let the solution cool while swirling every minute until the flask can be held for several seconds without unbearable heat 27. While hot, pipette in 10 µl ethidium bromide solution (EtBr,
0.5 mg/ml, used with caution as EtBr is a carcinogen and mutagen) and swirl until mixed
28. Pour into a horizontal gel rig and insert plastic combs holding ca. 10 µl of sample each
29. Let the gel solidify fully; gels can be wrapped in plastic wrap for longterm storage (overnight in place or for days at 4oC).
30. Mix 5 µl of the extraction solution with 2 µl of a 40% weight/ volume sucrose load buffer (made as 4 g sucrose and 25 mg bromophenol blue in 10 ml distilled water)
31. Submerge gel in a rig containing 0.5 x TBE, remove gel comb and load the 7 µl of sample/dye mix in separate wells using DNA molecular weight standards (e.g., 500 bp molecular ruler, www.biorad.com)
32. Draw the DNA across the gel toward the anode/positive charge at ca. 100 V depending on the gel rig size and specifications.
33. Monitor via the blue bromophenol blue stain movement (which tracks a DNA size fragment of ca. 300 bp in a 1.5% gel), stopping the gel and visualizing the DNA using ultraviolet light when it has progressed enough.
34. DNA can also be quantified via a spectrophotometer such as the Nanodrop (www.nanodrop.com), following
manufacturer’s protocol: Briefly, after calibration 1 µl of nucleic acid solution is placed onto a cleaned pedestal, the lid is closed and a reading is taken prior to cleaning by wiping the pedestal in preparation for the next sample. The machine will estimate concentration using the equation dsDNA: A260
1.0 = 50 ng/µl. 35. Store at -20°C or below.
3.2.2 DNA extraction using Qiagen Blood and Tissue DNA kits
This is a reliable extraction method using a commercial kit sold by Qiagen (www.quiagen.com), it is suitable for honey bee guts, small larvae or tissues from larger larvae or adults (avoid using the compound eyes).
1. Place 50 mg honey bee material in a centrifuge tube and mince thoroughly on ice with a mini pestle
2. Add 180 µl Buffer ATL and 20 µl Proteinase K at the provided concentration
3. Vortex 30 seconds and incubate at 56oC for 1 hour, vortexing
for 30 seconds after 30 min
4. Premix equal volumes of Buffer AL and ethanol (96-100%), mixing enough to provide 400 µl per sample plus 10% extra 5. Vortex samples 30 seconds and add 400 µl AL/EtOH mix each,
vortex again 30 seconds
6. Pipette all into DNeasy Mini spin column nested in a 2 ml collection tube.
7. Centrifuge at > 8000 rpm in a microcentrifuge (6k g). Discard flow-through and collection tube
8. Place spin column in new 2 ml collection tube, add 500 µl Buffer AW1, centrifuge 1 min at > 8000 rpm. Discard flow-through and collection tube
9. Place spin column in new 2 ml collection tube, add 500 µl Buffer AW2, centrifuge 3 min at > 14000 rpm. Discard flow-through and collection tube
10. Remove spin column, checking to be sure ethanol is gone and place into a clean 1.7 ml centrifuge tube
11. Add 200 µl Buffer AE to the centre of the membrane, incubate at room temperature and then centrifuge for 1 min at > 8000 rpm. Eluted DNA will be in tube. Check quantity by Nanodrop or agarose gel as in section 3.2.1 above.
3.2.3. DNA extraction using Chelex
The Chelex method (Walsh et al., 1991) provides a very rapid way to protect DNA from degradative enzymes and from some of the potential contaminants that might inhibit experiments. In principle, the Chelex resin will trap salts needed by degradative enzymes, leaving DNA in solution. In practice, Chelex extractions can be prone to degradation, and should be kept in the freezer when not in use, or these extractions should be used within 24 hours of extraction. If the extracted tissues contain pigments and other inhibitors for
downstream experiments, it is often successful to dilute the Chelex extraction 1:9 with distilled water before use. Finally, when drawing from these extractions it is important to pipette from the top of the aqueous layer, avoiding the resin itself. Below is a recipe that works well for legs from adult bees or beetles, for whole varroa mites, or for other tissues of about that size.
1. Add two posterior legs into Eppendorf tubes. 2. Allow them to dry until the EtOH evaporates. 3. Transfer to each tube:
100 µl of Chelex® (5% solution in water),
5 µl of proteinase K (10 mg/ml).
4. Incubate the samples in a thermocycler with the following program: 1 h at 55°C, 15 min at 99°C, 1 min at 37°C, 15 min at 99°C, Pause at 15°C.
3.3. DNA detection using southern blots with DIG
labelling
Southern blotting was invented by Edward M Southern as a means for detecting specific nucleotide sequences in a complex mixture and determining the size of the restriction fragments, which are complementary to a probe. Southern blotting combines transfer of restriction-enzyme-digested and then electrophoresis-separated DNA fragments from a gel to a membrane and subsequent detection by probe hybridization. A variety of non-radioactive methods have been developed to label probes for detection of specific nucleic acids. The Roche Applied Science DIG system is a simple adaptation of
enzymatic labelling and offers a non-radioactive approach for the safe and efficient labelling of probes for hybridization reactions.
3.3.1. Restriction enzyme digestion and agarose gel electrophoresis
This step is carried out in order to array chromosomal sections across a one-dimensional space so that unique sections can be probed for matches to a query sequence. In principle, the targeted gene will be embedded in a single chromosomal segment flanked by specific sequences that match the restriction enzyme used.
1. Digest 5-10 µg of genomic DNA in a volume of 30 µl with an appropriate restriction enzyme by setting up reaction as follows:
3 µl 10X buffer,
0.3 µl of BSA if needed (this will be on the restriction enzyme label),
3 µl enzyme (10U/µl), 5-10 µg genomic DNA,
Add sterile water to reach a total volume of 30 µl.
Generally, enzymes that cut frequently in the target genome are used here (e.g., ‘four-cutters’ that cut at a specific four-base-pair sequence, an event expected to occur ca. once every several hundred base-pairs).
2. Allow the digestive reaction to go for overnight at 37°C (or temperature appropriate to your specific enzyme).
3. Run the full 30 µl of reaction mixture with 3 µl 6X loading dye on 1% agarose gel (see section 3.2.1) containing ethidium bromide (1 µg/ml ) for 2 hours at 100 volts. Include one lane of a DIG-labelled DNA Molecular Weight Marker at the appropriate level.
4. Take a picture of the digestion.
5. Depurinate the agarose gel for exactly 10 min in 0.25 M HCl if DNA fragment > 4 kb.
6. Denature the gel in freshly made denaturing solution (0.5M NaOH, 1.5 M NaCl) for 2 x 15 min at RT, slowly shaking on rotating shaker.
Denaturation of the DNA into single strands allows hybridization with a probe possible.
7. Rinse the gel with sterile water.
8. Neutralize the gel in neutralizing solution (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl) for 2 x 15 min, slowly shaking on rotating shaker.
9. Equilibrate gel in 20X SSC for 10 min.
3.3.2. Assembly of the transfer setup and transfer of DNA from gel to membrane
DNA is here pulled from the gel into a nylon membrane by capillary action pulled by the positive charge of the membrane. Once in contact with the membrane, DNA is attached using high-voltage cross-linking.
1. Set up capillary transfer using 20X SSC as a transfer agent: Inside a baking glass dish filled with 20X SSC, place a glass plate that is elevated by four rubber stoppers that is slightly larger than the gel.
2. Cover the glass plate with a piece of wick-blotting paper that has to be long enough so that it is in contact with the 20X SSC transfer solution.
The buffer flows up the wick-blotting paper by capillary action, then through the gel to the membrane.
3. Smooth out the air bubbles between the glass and the blotting paper by gently rolling with a glass pipette. 4. Place the gel facing down on the wet blotting paper. 5. Cut a small triangular piece from the top left-hand corner to
simplify orientation. 6. Smooth out the air bubbles.
7. Cut one piece of positively charged nylon membrane to match size of the gel.
8. Soak the membrane in water for 2-3 min to wet and then float in 20X SSC.
9. Gently place the membrane on the top of the gel. 10. Mark well positions on the membrane.
11. Smooth out the air bubbles.
12. Cut 4-5 sheets of Whatman 3MM paper to the same size as the gel and place on top of the membrane.
13. Place a stack of paper towels on top of the Whatman 3MM papers.
14. Add a 200-400 g weight to hold everything in place. 15. Allow the DNA to transfer for 10-16 hours.
16. After transfer, rinse the membrane briefly in 6X SSC. 17. Immobilize DNA to the membrane by UV cross-linking
(120,000 microjoules per cm²). Membrane is now ready for labelling (section 3.3.3.).
3.3.3. Synthesis of DIG-labelled DNA probe
DIG-labelled probes offer a method to identify where probes have attached on the membrane (e.g.., the location of their targeted DNA match). These DIG probes are an alternative to highly regulated and more dangerous radio isotopic probes.
1. Mix the DIG-labelled PCR reaction components from the Roche Applied Science PCR DIG Labelling Mix with the probe template as follows:
5 µl PCR Buffer (10X), 5 µl PCR DIG Labelling Mix, 0.5 µl Upstream Primer (25 µM), 0.5 µl Downstream Primer (25 µM),
0.5-1 µl Template (plasmid DNA, 10-100 pg, or genomic DNA, 1-50 ng),
0.75 µl Enzyme Mix,
Add sterile water until total reaction volume is equal to 50 µl. 2. Set the annealing temperature of PCR reaction to reflect the
predicted annealing temperature of the primers, also reported at the time of purchase.
3. The kit contains a post-hoc check for probe labelling efficiency that is recommended.
3.3.4. Hybridizing the DIG-labelled DNA Probe to DNA on the Blot
This procedure relies on the Roche Applied Science DIG Easy® Hyb,
DIG Wash and Block Buffer Set, (with the fluorescent reporter CSPD®).
1. Pre-warm an appropriate volume of DIG Easy® Hyb solution®
to the hybridization temperature.
2. Pre-hybridize membrane in a small volume of pre-warmed DIG Easy® Hyb solution (20 ml if in a 200 ml hybridization
tube).
3. While the membrane is pre-hybridizing, denature 10 µl of DIG -labelled DNA by boiling for 5 min.
4. Rapidly cool on ice.
5. Add appropriate amount of denatured probe to give you (25 ng/ml) into DIG Easy® Hyb solution.
6. Incubate with agitation in a hybrid oven at 55-58oC for
overnight.
7. Wash membrane in 25-50 ml Washing Solution-1 (2X SSC, 0.1% SDS) 2X for 5 min at room temperature under constant agitation.
8. Wash membrane in 25-50 ml Washing Solution-2 (0.1% SSC, 0.1% SDS) 2X for 5 min at 68oC under constant agitation.
9. Wash membranes briefly (1-5 min) in 25 ml of 1X Washing Buffer provided in DIG Wash kit.
10. Incubate membranes for 30 min in 1X Blocking Solution diluted in maleic acid buffer (supplied in the kit). 11. Incubate membrane in Anti-body solution for 30 min. To make anti-body solution, add 1 µl anti-body to 20 ml 1X blocking solution.
12. Wash membrane in 1X Washing buffer 2X for 15 min. Make sure membrane is immersed in the Washing buffer.
13. Prepare 20 ml 1X Detection Buffer.
14. Equilibrate membrane in 20 ml 1X Detection Buffer for 2-5 min.
15. Transfer the membrane with DNA side facing up to a Plastic wrap that is at least twice the size of the membrane. 16. Apply 1 ml of CSPD®, ready-to-use (about 20-25 drops) to the
membrane.
17. Quickly cover the membrane with the plastic wrap. 18. Incubate for 5 min at RT.
19. Drain off excess buffer by gently brushing across the top of the membrane covered by plastic wrap with a paper towel. 20. Tape the membrane into a film cassette.
21. Close the cassette and incubate at 37°C for 10 min to enhance the luminescent reaction.
22. Remove the film for development using a standard x-ray film developer.
4. RNA methods
4.1. Introduction
Analyses based on RNA have two major advantages over DNA analyses. First, they are by definition restricted to a step in the expression of proteins from an organism’s genome. This means that RNA pools are generally far less complex than are pools of DNA representative of the organism’s entire genome, and that a
quantitative estimate of different RNA’s can provide a useful surrogate for the proteins produced at that time point for a specific organism or tissue within an organism. Second, since nearly all of the recognized viral threats to honey bee exist without a DNA stage, these threats are only visible via RNA analyses. These arguments make RNA the resource of choice for many honey bee analyses; despite greater concerns over storage and preservation of tissues.
A common strategy is to extract total nucleic acids directly in strongly denaturing buffers, so as to inactivate RNAses immediately during homogenisation. RNAses have numerous disulphide bridges. This makes them very stable in a very wide range of conditions, such that strong denaturants are required to permanently inactivate them. Heat, detergents (sodium dodecyl sulphate), organic solvents (phenol), proteinases, chaotropic salts (guanidine isothiocyanate), reducing agents (β-mercaptoethanol; dithiothreitol) and nucleic acid protecting compounds (CTAB; cetyl trimethylammonium bromide) are some of the more common methods used to inactivate RNAses. The nucleic acids can be purified from other compounds with affinity columns, magnetic bead-linked nucleic acid binding agents or by precipitation with alcohol or lithium chloride. The most common, quickest and most reliable combination is a chaotropic salt/β-mercaptoethanol extraction buffer, followed by purification on disposable affinity columns (Verheyden et al., 2003). The main disadvantage of RNA precipitation (with 2 volumes ethanol, 1 volume isopropanol or with 6M LiCl) is that many undesirable compounds often co-precipitate with the nucleic acid, requiring further precipitations or washes to clean the sample. There are many excellent commercial RNA extraction kits available, based on one or more of these principles. However, their performance in comparative tests varies greatly, depending on the organism, tissue type and nucleic acid extracted (Konomi et al., 2002; Knepp et al., 2003; Wilson et al., 2004; Schuurman et al., 2005; Labayru et al., 2005). Below are two protocols, representing the most common approaches to RNA extraction.
4.2. Affinity column purification
The processing consists of making a primary homogenate from 1-30 bees, purifying RNA from one (or more) aliquots of the homogenate using affinity columns, and measuring the RNA concentration. β-mercaptoethanol is toxic so processing should be done in a fume hood. Prepare all the buffers and tubes before starting.
The protocol below is based on the columns marketed by Qiagen or generic equivalents. The maximum recommended amount of tissue per column is 20 mg. More than 20 mg tissue causes the column to bind too much protein, reducing the yield and quality of the nucleic acid. Bees, pupae and large larvae weigh between 100-180 mg each, and so need to be homogenised first in a primary extract, from which a volume equivalent to 20 mg tissue is then processed on the affinity columns. A suitable denaturing buffer for this primary extract is a Guanidine Iso-Thio Cyanate (GITC) buffer, which has similar properties to the Qiagen RLT buffer:
1. Mix the GITC buffer:
5.25 M guanidinium thiocyanate (guanidine isothiocyanate), 50 mM TRIS.Cl(pH 6.4),
4.3.1 TRIzol® extraction
Advanced preparation: You will need RNase-free bench, pipettes, barrier tips, pestles and 1.5 ml microcentrifuge tubes. Bench tops and other glass and plastic surfaces can be treated to remove RNAse contamination by application of RNAse Zap (Ambion), following manufacturer’s protocol. Disposable tips, pestles, and microcentrifuge tubes should be purchased RNase–free. You will need cold 75-80% ethanol and 100% isopropanol, both nuclease-free; and a pre-chilled centrifuge (at 4°C for 30 min) for Step 9. Have ready at room temperature, the TRIzol® and other reagents needed. It is recommended
to use a vented fume hood for safety when working with TRIzol® and
chloroform. It is also very important to work quickly with bee tissue, as it is possible that RNA will degrade if bees thaw for ten or more minutes (Dainat et al., 2011).
In a very sterile (RNAase-free) environment:
1. Add 500 µl of TRIzol® to frozen bee abdomens in 1.5 ml
tubes.
2. Mash the tissue until completely homogenized with a pestle and shaking.
All soft tissues should be disrupted completely. Remove pestle and scrape it off along the rim of the microcentrifuge tube. Sample should be viscous.
3. Centrifuge at 5,000 rpm for 1 min to pellet debris.
4. Transfer the TRIzol® suspension to a fresh tube, leaving out
the chitinous debris pellet.
5. Add another 500 µl TRIzol® and invert several times to mix.
6. Add 200 µl chloroform. 7. Shake vigorously for 15 sec.
Do not vortex! This may increase DNA contamination in your sample. 8. Incubate at RT for 2-3 min.
9. Spin at 4°C for 15 min at ~14,000 rpm.
NOTE: Be especially diligent about avoiding RNases from this point on!
10. Label a fresh set of RNase-free microcentrifuge tubes. 11. Carefully remove tubes from centrifuge.
12. Use a 1 ml pipette tip with pipettor set at 550 µl to draw off the upper phase and transfer it to a fresh tube.
Carefully avoid the interface (one product that ensures a clean break between phases is the Phaselock gel (5 Prime Inc.) and could be used here).
13. Add 500 µl 100% Isopropanol. 14. Invert 3-5 times to mix gently. 15. Incubate at RT for 10 min.
16. Centrifuge at 4°C for 10 min at full speed (~12,500rpm), placing all tubes in the same rotation (e.g., hinge facing away from arc) so pellet location will be consistent.
1.3% Triton X-100, 1% β-mercaptoethanol.
2. Place exact, pre-determined number of frozen bees in the homogenizer of choice.
3. Per bee, add the following amount of GITC buffer:
With these extract volumes, 100 μl extract is approximately 20 mg bee tissue
4. Mix:
100 μl bee extract,
350 μl RLT buffer + 1% β-mercaptoethanol.
5. Proceed according to the Plant RNA extraction protocol (see Qiagen instructions booklet).
Inclusion of the Qia-shredder column step is not required, but significantly increases yield and purity of nucleic acid.
6. Elute in 100 μl nuclease-free water.
7. Determine nucleic acid concentration and purity (see section 8.4.; “Nucleic Acid Quality Assessment”).
8. Store as two separate 50 μl aliquots at -80oC, one for working
with and one for storage.
9. Include a ‘blank’ extraction (i.e. an extraction of purified water) after every 24 bee samples, to make sure none of the extraction reagents have become contaminated.
4.3. Acid phenol RNA extraction from adult bees
The below recipes use an acid-phenol phase separation for isolating RNA from DNA and other tissue components. The TRIzol® (Invitrogen™)
protocol is the most commonly used, and widely available, method of acid-phenol extraction of RNAs. However, using a generic lysis and acid-phenol buffer (e.g. section 4.3.2) provides a cost effective alternative than TRIzol®, and allows a great reduction in the use of
the caustic chemical phenol for pooled samples. We use honey bee abdomens because they provide representation of the microbes and immune components of the honey bee, while avoiding pigments in the eye which can inhibit downstream enzymatic reactions. The procedure is also appropriate for larvae, whole adult bees and pupal RNA extractions, if volumes are scaled upward, i.e. doubled, to reflect the volume of the sample, for the latter two.
Bee Weight Buffer Total volume
Worker bee 120 mg 500 μl 600 μl
Drone 180 mg 700 μl 900 μl
Worker pupa 160 mg 650 μl 800 μl Drone pupa 240 mg 1000 μl 1200 μl
